Site-specific recombination provides a vehicle to introduce exogenous DNA, delete DNA, or rearrange DNA at specific sites in a chromosome (41). Among the site-specific recombination systems characterized to date, the FLP system of the yeast 2 micron plasmid and the Cre-lox system of bacteriophage P1 are among the most attractive for genomic manipulation because of their efficiency, simplicity, and demonstrated in vivo activity in a wide range of organisms. These systems have been used to construct specific genomic deletions and gene duplications, study gene function, promote chromosomal translocations, promote site-specific chromosome cleavage, and facilitate the construction of genomic libraries in organisms including bacteria, yeast, insects, plants, mice, and humans 2-5, 10-18, 24-26, 28, 30-35, 38-41, 44, 45, 47, 50). These studies have only begun to tap the potential of the approach.
Site-specific recombination catalyzed by the FLP and Cre recombinases occurs readily in bacterial cells (1, 5, 6, 21, 33). In principle, it could find wide application to studies of genomic structure and function as well as enhance the usefulness of E. coli in biotechnology. Ironically, this approach has not been exploited in bacteria as it has been in eukaryotes, although bacteria were the first non-yeast cells in which FLP-mediated recombination was demonstrated (6). Even though gene targeting in bacteria can be achieved by homologous recombination, chromosomal targeting by site-specific recombination provides a new route to stable transformation with the advantages of very high efficiency, defined reproducible insertion sites in the chromosome, and controlled reversibility.
The yeast FLP system has been studied intensively (7, 8, 22, 36). The only requirements for FLP recombination are the FLP protein and the FLP Recombination Target (FRT) sites on the DNA substrates. The minimal functional FRT site contains only 34 bp. The FLP protein can promote both inter- and intra-molecular recombination.
Previously, the inventors (Huang, et al., 1991) reported the construction of a model system in E. coli using the FLP recombination system for chromosomal targeting and demonstrated the effectiveness of the general approach (21). The site-specific integration was absolutely dependent upon the expression of FLP protein and the presence of FRT sites in the chromosome. In some experiments, from 1% to 10% of the exogenous DNA molecules used, introduced on a modified bacteriophage .lambda. vector, actually found their way into a cell and were integrated into the chromosome specifically at a chromosomal FRT.
Although Huang, et al. (1991) achieved a high integration frequency in this original targeting system, there were limitations inherent to the constructs that precluded a detailed characterization as well as a convenient application of the system to bacterial cloning and genomic studies.